PL220536B1 - Certain amionicalcylivglucosaminiumphosphate compounds and their application - Google Patents

Abstract

Compounds that are adjuvants and immunoeffectors are described and claimed. The compounds increase the production of immunoeffectors. The compounds increase the production of antibodies in immunoimmunized animals and stimulate the production of cytokines and activate macrophages. Compositions and methods of using the compounds as adjuvants and immunoeffectors are also described.

Description

The present invention relates to an aminoalkyl glucosaminide phosphate compound.

Toll-type receptors (TLRs) have been associated with a strong innate immune response and recognize various structural components that are unique to pathogens. Such an interaction brings the immune system into an active state with short and long-term effects. There is considerable interest in the development of TLR agonists and antagonists as pharmacological manipulation of innate immune responses could lead to more effective vaccines and a new therapeutic approach to autoimmune, atopic, malignant and infectious diseases. The first microbial product found to be a Toll receptor agonist was LPS, a gram-negative bacterial membrane component that activates the Toll receptor 4 (TLR-4). Although LPS is a potent immunomodulatory agent, its medical use is limited because of its extreme toxicity, including the induction of systemic inflammatory response syndrome. The biologically active, endotoxic, substructural moiety of LPS is lipid A, a phosphorylated polyacylated fatty acid glucosaminodisaccharide that serves to anchor the entire structure to the outer membrane of gram-negative bacteria. The toxic effects of lipid A can be alleviated by selectively chemically modifying lipid A to form monophosphorylated lipid A compounds (MPL ™ immunostimulant, Corixa Corporation, Seattle, WA). Methods of making and using the immunostimulatory agent MPL ™ and structurally similar compounds in a vaccine adjuvant as well as other uses have been described (see, for example, U.S. Patent Nos. 4,436,727, 4,877,611,4,866,034 and 4,912,094, 4,987,237, Johnson et al., J Med Chem 42: 46404649 (1999), Ulrich and Myers, Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, editors, Plenum: New York, 495-524, 1995). In particular, these and other references demonstrated that the MPL ™ immunostimulant and similar compounds showed significant activity as adjuvants when used in vaccine formulations with protein and carbohydrate antigens to increase humoral and / or cell mediated immunity to antigens and interacted with Toll type receptors.

Based on the experience with the immunostimulant MPL ™ and other components of the bacterial cell wall, a family of new synthetic compounds, aminoalkyl glucosaminide phosphates (AGP), was developed. AGP compounds also interact with TLR-4 as agonists and antagonists. AGPs include both acyclic and cyclic compounds (US Patent Nos. 6,113,918 and 6,303,347, Patent Application No. WO 98/50399 published October 12, 1998, WO 01/34617 published May 17, 2001, WO 01/90129 published November 29, 2001 and WO 02/12258 published February 14, 2002). Like the MPL ™ immunostimulant, these compounds have been shown to retain significant adjuvant properties when formulated with antigens in vaccine compositions, and further exhibit similar or better toxicity profiles as compared to MPL ™ immunostimulants. AGPs also exhibit mucosal adjuvant activity and are effective also in the absence of antigen, which makes them interesting compounds for prophylactic and / or therapeutic applications.

Another significant advantage offered by AGP over the immunostimulant MPL ™, etc., is that the AGP can be easily produced on an industrial scale by synthetic methods. Because they are produced synthetically, the AGPs are free from the trace biological contaminants found in MPL ™. As such, AGPs have advantages over MPL as vaccine adjuvants in certain systems, such as pediatric immunization protocols, where pyrogenicity of the adjuvant must be minimized. However, since AGP is synthesized chemically, a less than optimal compound stability can lead to an accumulation of degradation products which can result in variable biological activity and batch-to-batch stability. From a point of view of developing GMP processes in the production of materials for human clinical trials, the main challenges are batch stability and batch-to-batch stability variability. Therefore, compounds are desired that exhibit greater biological activity as compared to the immunostimulant MPL ™, etc., interact with Toll-type receptors, and / or are optimal for large-scale synthesis of GPL. The present invention addresses these needs and even more so to provide compounds modified for greater biological activity, stability with increased resistance to enzymatic and chemical degradation, and / or better safety profiles.

PL 220 536 B1

The present invention relates to a compound having the formula:

where n is 1 or 5 and R6 is COOH or CH2OPO3H2.

Preferably n is 1.

Preferably n is 5.

Preferably R6 is COOH.

Compounds of the present invention are members of the aminoalkylglucosaminide-4-phosphate (AGP) family. As described below, the compounds described herein have hexa-acyl chain length variable (primary and secondary) modifications, alkyl branch structural modifications including a phosphate moiety, a structural modification involving the primary ether lipid at the C-3 position of the sugar, and three secondary etherolipids and / or the 6-hydroxyl group. blocking.

Chemically known as ro-aminoalkyl-2-amino-2-deoxy-4-phosphono-pD-glucopyranosides, AGPs are a class of synthetic lipid A mimetics that are structurally related to the major biologically active ingredient component in monophosphoryl lipid A. In AGP, reducing sugar has been replaced by an N - [(R) -3-n-alkanoyloxytetradecanoyl] aminoalkyl aglycone unit. As with other lipid A disaccharides, AGPs contain six fatty acids for maximum biological activity, and unlike disaccharides, AGPs contain a conformationally flexible β-linked aglycone unit which allows energetically privileged tight packing of the six fatty acid chains. The tight packing of the six fatty acids in the hexagonal arrangement is believed to play an essential role in the biological activity of lipid A-type molecules (Seydel et al., Immunobiol, 187 (3-5): 191-211, 1993).

The compounds of the present invention are considered members of the AGP family. These compounds contain modifications to the length of the six acyl chains (primary and secondary).

In one of the broadest aspects, an AGP compound of formula (I) is described herein:

Wherein X is selected from the group consisting of O and S in an axial or equatorial position, Y is selected from the group consisting of O and NH, n, m, p and q are integers from 0 to 6, R1, R2 and R3 are the same or different and represent fatty acid acyl residues containing from 1 to about 20 carbon atoms, wherein one of R1, R2 or R3 is optionally hydrogen, R4 and R5 are the same or different and are selected from the group consisting of H and methyl, R6 and R7 are the same or different and are selected from the group consisting of H, hydroxy, alkoxy, phosphono, phosphonooxy, sulfonate, sulfoxy, amino, mercapto, cyano, nitro, formyl and carboxylic acid, and its esters and amides, R8 and R9 are the same or different and are selected from the group consisting of a phosphono group and H, wherein at least one of R8 and R9 is a phosphono group, R10, R11 and R12 are independently selected from simple, unsubstituted, saturated aliphatic groups having from 1 to 10 atoms coal, or his pharmacist totally acceptable salt.

In preferred embodiments of this aspect

- X and Y are preferably oxygen,

- R ₁ , R ₂ and R ₃ are preferably normal acyl groups and especially are independently selected from simple C 6 -C 10 acyl groups (in particular saturated acyl groups),

- R10, R11 and R12 are preferably unsubstituted, saturated aliphatic (i.e. alkyl) groups having from 1 to 10, preferably from 3 to 9, especially from 3 to 7 carbon atoms, and especially are identical unsubstituted, saturated aliphatic groups containing from 3 to 7 carbon atoms.

Compounds 1a, b and 2a, b and their pharmaceutically acceptable salts are examples of this type of compound (I).

Compounds (I) thus resemble certain known AGPs except that they have shorter primary fatty acid chains. It has been found that altering the length of the secondary fatty acid chain has an effect on the immune stimulating ability of AGP, and in the case of secondary fatty acid homologues, 3-D-MPL (Johnson et al., J Med Chem, 42: 4640-4649, 1999). The low endotoxicity of some natural lipid A variants, such as lipid A from R. sphaeroides, has been attributed in part to the presence of shorter (C10) primary fatty acids in these molecules (Qureshi et al., J Biol Chem, 266 (10): 6532-6538, 1991 ). Similarly, the low toxicity of some LPS from helicobacteria and pseudomonas may be due to the presence of hexacyl components containing primary fatty acids that differ in length from the acids found in the toxic lipid A of salmonella (Moran et al., J Bacteriol, 179 (20): 6453-6463, 1997, Kulshin et al., Eur J Biochem, 198 (3): 697-704, 1991). Although the relationship between the length of the primary acyl chains has been studied to a limited extent with synthetic analogs of lipid A subunits containing three fatty acids (Hasegawa et al., Biosci Biotech Biochem, 59 (9): 1790-1792, 1995, and Ogawa et al., Carbohydr Res, 220 : 155-164, 1991) and tetraacyl disaccharide analogs of lipid IVa (Fukase et al., Tetrahedron, 54: 4033-4050, 1998), to our knowledge no systematic studies have ever been performed with either the basic hexacylated pharmacophore of lipid A or the lipid mimetic AND.

Certain glycyl and phosphonooxyethyl (PE) compounds (L-serinol phosphates) also belong to the group of compounds described herein. These are compounds of the above formula (I), in which R5 and R7 are hydrogen, n, m and q are 0, p is 1 and R6 is COOH, or in which p is 2 and R6 is

OPO3H2. Thus, they have the general formula (II):

PL 220 536 B1

wherein X is selected from the group consisting of O and S in an axial or equatorial position, Y is selected from the group consisting of O and NH, n and m are 0, R1, R2 and R3 are the same or different and represent acyl residues of fatty acids containing from 1 up to about 20 carbon atoms, wherein one of R1, R2 or R3 is optionally hydrogen, R4 is selected from the group consisting of H and methyl, p is 1 and R6 is COOH, or p is 2 and R6 is OPO3H2, R8, and R9 are the same or different and are selected from the group consisting of a phosphono group and H, wherein at least one of R8 and R9 is a phosphono group, and R10, R11 and R12 are independently selected from simple, unsubstituted, saturated aliphatic groups containing from 1 up to 10 carbon atoms, or a pharmaceutically acceptable salt thereof.

In the preferred embodiments of compounds (II)

- X and Y are preferably oxygen,

- R ₁ , R ₂ and R ₃ are preferably normal acyl groups and especially are independently selected from simple C 6 -C 10 acyl groups,

- the groups R10, R11 and R12 are preferably unsubstituted, saturated aliphatic (i.e. alkyl) groups having from 1 to 10, preferably from 3 to 9, especially from 3 to 7 carbon atoms, and in particular represent identical, unsubstituted, saturated aliphatic groups containing from 3 to 7 carbon atoms.

Examples of this type of compound (II) are compounds 11a, b and 12a, b.

IIa R ₂ = Ceacyl IIlb R _z = Cioacyl

12a R ₂ = Qacyi 12b R ₂ = Cio3Cyi

Compounds 12a, 12b contain structural modifications to the alkyl branch containing a phosphate moiety. Such relationships are believed to be potentially more permanent than other family members. These compounds have an advantage over the AGP seryl / serinol phosphate classes in that they do not contain a stereogenic center in the aglycone moiety, a feature that can complicate synthesis and lead to difficulties in separating enantiomeric or diastereomeric impurities.

PL 220 536 B1

Another type of compound described herein is the (R) -3-alkyloxytetradecanoic acid derivatives. These derivatives have the same general formula (II) above except that R1, R2 and R3 are not acyl groups but are simple alkyl groups forming the groups R1O-, R2O- and R3O- rather than carboxylic acid derivatives. In this type of compound, R1, R2 and R3 are preferably C6-C10-alkyl groups which may be the same or different, but most preferably identical.

Such compounds have the general formula (III):

wherein X is selected from the group consisting of O and S in an axial or equatorial position, Y is selected from the group consisting of O and NH, n and m are 0, R1, R2 and R3 are the same or different and represent acyl residues of fatty acids containing from 1 up to about 20 carbon atoms, wherein one of R1, R2 or R3 is optionally hydrogen, R4 is selected from the group consisting of H and methyl, p is 1 and R6 is COOH, or p is 2 and R6 is OPO3H2, R8, and R9 are the same or different and are selected from the group consisting of a phosphono group and H, wherein at least one of R8 and R9 is a phosphono group, and R10, R11 and R12 are independently selected from simple, unsubstituted, saturated aliphatic groups containing from 1 up to 11 carbon atoms, or a pharmaceutically acceptable salt thereof.

In the preferred embodiments of compounds (III)

- X and Y are preferably oxygen,

- R _1, R ₂ and R ₃ are preferably independently selected from unsubstituted, straight C6-C10 alkyl,

- the groups R10, R11 and R12 are preferably unsubstituted, saturated aliphatic (i.e. alkyl) groups having from 1 to 11, preferably from 3 to 9, especially from 3 to 7 carbon atoms, in particular identical, unsubstituted, saturated aliphatic groups containing from 3 to 7 carbon atoms.

Compounds 18a, b are examples of this group, containing a primary ether lipid in the C-3 position of the sugar and three secondary ether lipids

ifia n = i (Ce alkyl)

186 n «5 (Cioilktfl R _{1 S} = CH ₂ OPO ₃ H 2 or CO 2H

PL 220 536 B1

Yet another type of compound described here has formula (IV):

ORp

R, O

ABOUT

_OR3 (IV) wherein Y is oxygen, X is selected from the group consisting of O and S at the position of an axial or equatorial, n and m are 0, R _1, R ₂ and R ₃ are the same or different and represent acyl residues of fatty acids from 1 to about 20 carbon atoms, wherein one of R _1, R ₂ or R ₃ is optionally hydrogen, R ₄ is selected from the group consisting of H and methyl, p is 1 and _R6 is COOH or p is 2, and R ₆ is OPO ₃ H ₂ , R ₈ and R ₉ are the same or different and are selected from the group consisting of a phosphono group and H, with at least one of R ₈ and R ₉ being a phosphono group and R ₁₀ , R 11 and R12 are independently selected from simple unsubstituted saturated aliphatic groups having from 1 to 10 carbon atoms, or a pharmaceutically acceptable salt thereof.

These compounds therefore have two acylated chains and one non-acylated ether chain.

In the preferred embodiments of compounds (IV)

- X is preferably O,

- R _1, R ₂ and R ₃ are most preferably selected from unsubstituted straight C6-C10 alkyl,

- the groups R ₁₀ , R ₁₁ and R ₁₂ are preferably unsubstituted, saturated aliphatic (i.e. alkyl) groups having from 1 to 10, preferably from 3 to 9, especially from 3 to 7 carbon atoms, especially identical, unsubstituted, saturated aliphatic groups containing from 3 to 7 carbon atoms.

20a, b are examples of this class of compounds.

20a n-1 (C _S alkyl! 20b n => 5 (C 10 alkyl)

Ri = CHiOPQsH2 or COjH

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These compounds have features that allow resistance to unfavorable metabolism and / or aqueous hydrolysis. It is postulated that the selective removal of normal fatty acids in structurally different lipid A molecules by human acyloxyacyl hydrolase (AOAH) to obtain a lipid IV antagonist has developed as a defense mechanism to reduce lipid A toxicity (Erwin and Munford, J Biol Chem 265 ( 27): 15444-16449,1990). However, the greater toxicity of naturally derived 3-D-MPL relative to that of the major hexaacyl component is probably due to the presence of less highly acylated components with structures different to that of the IVa lipid (Ulrich and Myers, Monophosphoryl lipid A as an Adjuvant. Past experiences and new directions. In: Vaccine Design: The Subunit and Adjuvant Approach, eds. Powell MF, Newman MJ Plenum Press, New York, 1995, pp. 495-524, Johnson et al., J Med Chem, 42: 4640-4 649, 1999). Structural variability in 3-D-MPL and other lipid A preparations results intrinsically from related LPS and from ester cleavage during the semi-synthesis and isolation procedures. In fact, it has been reported that easy hydrolytic cleavage of the ester-bound acyl groups during chemical synthesis of putative lipid A from R. capsulatus, a potent antagonist of LPS-induced TNF-α production, results in lower amounts of undesirable agonist by-products (Christ et al., Science, 268). : 80-83,1995). Thus, chemical and / or enzymatic instability may be the Achilles heel of a potential lipid A drug containing ester labile bonds. The chemical and metabolic instability of ester-bound fatty acids present in both lipid A agonist and antagonist molecules has been resolved with hydrolytically stable analogs containing other linkages in place of primary and / or secondary ester-linked fatty acids (Christ et al., See above, Lien et al., J Biol Chem, 276 (3): 1873-1880, 2001).

Other compounds in the group described herein have a 6-hydroxy blocking group. These compounds have the formula (V):

wherein X is selected from the group consisting of O and S at the position of an axial or equatorial, Y is selected from the group consisting of O and NH; n, m, p and q are integers from 0 to 6, R _1, R ₂ and R ₃ are the same or different and represent acyl residues of fatty acids containing from 1 to about 20 carbon atoms, wherein one of R _1, R ₂ or R ₃ is optionally hydrogen, R ₄ and R ₅ are the same or different and are selected from the group consisting of H and methyl, R ₆ and R ₇ are the same or different and are selected from the group consisting of H, hydroxy, alkoxy, phosphono, phosphonooxy, sulfone, sulfoxy, amino, mercapto, cyano, nitro, formyl and carboxylic acid and esters thereof and amides, R 8 represents a phosphono group, PG is a hydroxy protecting group, as defined below, and R10, R11 and _R12 are independently selected from linear, unsubstituted saturated aliphatic groups having from 1 to 10 carbon atoms, or a pharmaceutically acceptable salt thereof .

The term "protecting group" (referred to herein as "PG") refers to a large number of groups used to replace the hydrogen of a hydroxyl group so as to block, prevent, or reduce group reactivity. Examples of protecting groups (and a list of commonly used abbreviations for them) can be found in T.W. Greene and P.G. Futs "Protective Groups in Organic Chemistry" (Wiley), Beaucage and Iyer, Tetrahedron 48: 2223, and Harrison et al., Compendium of Synthetic Organic Chemistry, Volumes 1-8 (Wiley). Representative protecting groups for hydroxyl groups include those in which the hydroxy group is either arylated or alkylated, such as in the formation of ethers or esters, using, for example, methyl, acetyl, benzyl, trityl, alkyl, tetrahydropyranyl, allyl, and trisubstituted silyl groups. or in which the hydroxyl group is replaced by fluorine.

The choice of protecting group for a given compound, target or set of conditions is within the skill of the art and is done in such a way as to protect, generally or selectively, a given reactive group under the prevailing conditions (presence of other reactive groups, pH, temperature , etc.). Protecting groups that can be used with the compounds described herein include methyl, phthaloyl, acetyl (Ac), benzyl (Bn), 2,2,2-trichloroethoxycarbonyl (Troc), t-butyldimethylsilyl (TBS), t-butyldiphenylsilyl (TBDPS) ) and 2,2,2-trichloro-1,1-dimethylethylchloroformyl (TCBOC). A fluorine atom can also be used as a protecting group. As is known in the art, certain protecting groups or types of protecting groups may be more suitable than others for use with a particular compound or situation, and take advantage of these properties when developing methods involving compounds with reactive groups such as a hydroxyl group.

In the preferred embodiments of these compounds (V)

- X and Y are preferably oxygen,

- R ₁ , R ₂ and R ₃ are preferably normal acyl groups and especially are independently selected from simple C 6 -C 10 acyl groups,

- the groups R ₁₀ , R ₁₁ and R ₁₂ are preferably unsubstituted, saturated aliphatic (i.e. alkyl) groups having from 1 to 10, preferably from 3 to 9, especially from 3 to 7 carbon atoms, especially identical, unsubstituted, saturated aliphatic groups containing from 3 to 7 carbon atoms.

Examples of this group include compounds 25a, b which have a methyl ether or compounds 26a, b which have a fluoro group used in conjunction with AGP seryl or serinol phosphate.

The unprotected hydroxyl group of the C6-sugar can lead to small amounts of impurities during the synthesis of lipid A derivatives that can be difficult to remove (Christ, see above). These by-products are likely formed from the initial formation of 4,6-cyclic phosphate followed by rearrangement (Imoto et al., Tetrahedron Lett. 29 (28): 2227-2230, 1988).

As already discussed herein, the term "aliphatic" as such or as part of another substituent means, unless otherwise stated, a straight or branched chain or cyclic hydrocarbon radical or combinations thereof, which may be fully saturated, mono- or polyunsaturated and may be include divalent and polyvalent radicals and has the specified number of carbon atoms (for example, C1-C10 is from one to ten carbon atoms). Examples of saturated hydrocarbon radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, cyclohexyl-methyl, cyclopropylmethyl, homologues of the isomers e.g. heptyl, n-octyl, etc. An unsaturated aliphatic group is a group that has one or more double bonds or triple bonds. Examples of unsaturated aliphatic groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3- butynyl and higher homologues and isomers. The aliphatic group will typically be from 1 to

Carbon atoms. A "lower" aliphatic group is a shorter chain aliphatic group and generally has eight or fewer carbon atoms.

The term "acyl" refers to a group derived from an organic acid by removal of the hydroxyl group. Examples of acyl groups include acetyl, propionyl, dodecanoyl, tetradecanoyl, isobutyryl, etc. Accordingly, the term "acyl" as used herein is understood to include a group otherwise termed -C (O) -aliphatic, in which the aliphatic group is preferably a saturated group aliphatic.

It is understood that the term "pharmaceutically acceptable salts" includes salts of the active compounds which are obtained with relatively nontoxic acids or bases depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include a sodium, potassium, calcium, ammonium, organic, amine or magnesium salt, or the like. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrocarbyl, phosphoric, monohydrophosphoric, dihydrogenphosphoric, sulfuric, monohydrogen sulfur, hydroiodic or phosphorous acid etc. organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, cork, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, etc. salts also include , such as arginate, and the like, and salts of organic acids such as glucuronic or galacturonic acid, and the like (see, for example, Berge, SM, et al. "Pharmaceutical Salts", Journal of Pharmaceutical Science, 66, 1-19, 1977). Some of the specific compounds described herein contain both basic and acid functionalities which allow the compounds to be converted into both base and acid addition salts.

The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of this invention.

In addition to the salt forms of the compounds described herein, compounds have been developed that exist in prodrug form. Prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions to yield the compounds of the present invention. In addition, pro-drugs can be converted into the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. Prodrugs can be slowly converted to, for example, the compounds of the present invention when placed in a transdermal patch reservoir with an appropriate enzyme or chemical reagent.

Some of the compounds described herein can exist in unsolvated forms and in solvated forms, including hydrated forms. The solvated forms are generally equivalent to the unsolvated forms. Some compounds may exist in multiple crystalline or amorphous forms. For the uses considered in the present invention, all physical forms are equivalent.

Some of the compounds described herein have asymmetric carbon atoms (optical centers) or double bonds and can then exist as racemates, diastereoisomers, geometric isomers, and individual isomers.

The compounds described herein may also have unnatural proportions of isotopes of atoms on one or more of the atoms that make up those compounds. The compounds can be, for example, radiolabeled with radionuclides such as for example tritium ⁽³ H), iodine-125 ⁽¹²⁵ I) or carbon-14 ⁽¹⁴ C).

The compounds of the present invention can be prepared by any available means (see, for example, section below), many of which have been described. Methods for the preparation of certain compounds useful in the present invention are known, for example, from US Patent Nos. 6,113,918, 6,303,347 and from PCT / US98 / 09385 (WO 98/50300 filed October 12, 1998). Still other compounds can be prepared by methods outlined by Johnson et al.

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J. Med. Chem. 42: 4660-4649 (1999), Johnson et al. Bioorg. Med. Chem. Lett. 9: 2273-2278 (1999) and in PCT / US98 / 50399 (WO 98/50399 published November 12, 1998). In general, the synthetic methods described in the aforementioned references, or other synthetic methods known in the art, are widely used in the preparation of these compounds. For example, in preparing compounds having different acyl groups and substituents, one skilled in the art will appreciate that the convergent methods described herein may be modified using other acylating agents, or may be initiated with commercially available materials which have the appropriate acyl groups attached.

In compositions for inducing or enhancing an immune response, the compounds of the subject invention are administered to warm blooded animals, including humans, with an antigen, such as a protein or polypeptide antigen, or with a polynucleotide that expresses the protein or polypeptide antigen. The amount of antigen administered to elicit the desired response can be readily determined by one of ordinary skill in the art, and will vary with the type of antigen administered, the route of administration, and the immunization schedule.

The compounds of the present invention may also be administered in the absence of an exogenous antigen to elicit immediate protection through the effect of nonspecific immunity, as described below (see Persing et al., WIPO publication WO 01/90129 dated November 29, 2001). Compounds that are capable of inducing non-specific immunity and / or of producing an adjuvant effect can be used in the rapid formulation of a vaccine. Administration of the compounds of the present invention with an antigen results in an acquired mucosal immune response within three to four weeks. Weekly administration of such compounds, for example by the intranasal route, over a period of four weeks should provide rapid and sustained protection by combining the protection provided by the initial innate immune response followed by the acquired immune response to the antigen of interest.

The compounds of the present invention can be evaluated using a variety of assay formats in order to identify and select those compounds having the characteristics best suited to a particular use of the invention. For example, animal models can be used to identify and evaluate cytokine secretion profiles into the circulation following administration of a compound of the present invention. In addition, there are various in vivo and in vitro models to study changes in one or more aspects of the immune response to different antigenic components in order to identify compounds best suited to elicit the specific immune response of interest. For example, the compound can be contacted with target cells such as macrophages, dendritic cells or Langerhans cells in vitro and the resultant cytokines can be measured. In addition, gene expression systems can be used to identify specific pathways activated or inhibited by a particular compound of interest.

Cytokine production / induction can be determined by treating human blood and / or cells with the compounds of the present invention and measuring the magnitude of the induction with an ELISA assay (R&D Systems). Such methods can also be used to determine if the development is dependent on the Toll receptor. The cytotoxic response to T cells following administration of the compounds of the present invention is determined by a ⁵¹ Cr-based cytotoxicity assay. If desired, the efficacy of a compound of the invention can be compared in this respect with other compounds known in this respect as functional compounds such as lipid A, MPL, AGP, etc. In addition, the compounds of the invention can be assessed in combination with one or more an adjuvant and / or immunostimulating agent for identifying synergistic effects (see, for example, US Patent Nos. 6,303,347 and 6,113,918 and WO 01/90129 published November 29, 2001).

Animal models, such as the rodent influenza induction model and the rodent Listeria monocytogenes induction model, are useful for assessing the activity of an adjuvant and an immunostimulatory agent. Briefly, the compound is administered after induction of influenza or L. monocytogenes. Disease index (ruffled fur, hunched posture and heavy breathing), weight loss and mortality, in the case of influenza, or the number of colony forming units in the spleens of treated / untreated mice, are monitored in L. monocytogenes as an indicator of protection provided by the administration of compounds according to of the invention (see, for example, WO 01/90129 published November 29, 2001).

The term "polypeptide" as used herein is used in its conventional sense, that is, an amino acid sequence. Polypeptides are not limited to a specific product length, and thus the term polypeptide includes peptides, oligopeptides and proteins, and unless otherwise indicated, such terms may be used interchangeably. This term, therefore, does not refer to or exclude modification of the polypeptide upon expression, e.g., glycosylation, acetylation, phosphorylation, etc.

And other modifications known in the art, both naturally and unnaturally occurring. The polypeptide can be the entire protein or a sub-sequence thereof. Polypeptides of particular interest in the context of the present invention are epitope-containing amino acid sequences, that is, antigenic determinants responsible to a large extent for the immunogenic properties of the polypeptide and capable of eliciting an immune response.

Polypeptides useful in the present invention are sometimes referred to herein as tumor proteins or tumor polypeptides as an indication that their identification has been based at least in part on their higher expression levels in tumor samples. Thus, "neoplastic polypeptide" or "neoplastic protein" generally refers to a polypeptide sequence or a polynucleotide sequence encoding such a polypeptide which expresses a significant proportion of the neoplastic sample, for example especially greater than about 20%, especially greater than about 30%, and especially greater than about 50% or more in the tumor samples tested, at a level that is at least twice, and preferably at least five times, the expression level in normal tissues as determined using a representative assay provided herein.

In some preferred embodiments, the above-mentioned polypeptides are immunogenic, i.e. they react detectably in immunological assays (such as an ELISA or T-cell stimulation assay) with immune sera and / or T cells from a cancer patient. Searching for immunogenic activity can be performed using techniques well known to one skilled in the art, and so, for example, such searches can be performed using methods such as those described by Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, the polypeptide can be immobilized on a solid support and contacted with patient sera allowing antibodies in the sera to bind to the immobilized polypeptide. Unbound sera can then be discarded,

Detect bound antibodies using, for example, ¹²⁵ I-labeled protein A.

As one skilled in the art would recognize, the polypeptides described herein also include the immunogenic portions of those polypeptides. As used herein, an "immunogenic portion" is a fragment of an immunogenic polypeptide that itself immunologically (ie, binds specifically) with B cells and / or with surface T-cell antigen receptors that recognize the polypeptide. In general, immunogenic portions can be identified using well-known techniques, such as those collected by Paul, Fundamental Immnunology, 3rd edition, 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening for polypeptides for their ability to react with antigen-specific antibodies, sera, and / or T cell lines or clones. Antibodies and antibodies as used herein are "antigen-specific" if they bind specifically to a protein (that is, they react with the protein in an ELISA or other immunoassay, and do not detectably react with unrelated proteins). Such antisera and antibodies can be prepared as described herein and using well known techniques.

In one preferred embodiment, the immunogenic portion of a polypeptide described herein is a portion that reacts with immune sera and / or T cells at a level that is not substantially less than the reactivity of the full-length polypeptide (e.g., in an ELISA assay and in a T-cell reactivity assay). . More particularly, the level of the immunogenic activity of the immunogenic portion is at least about 50%, especially at least about 70%, and more preferably greater than about 90% of the immunogenicity of the full length polypeptide. Under certain conditions, preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, that is, an activity level greater than about 100% or 150% or more of the immunogenic activity.

In some other embodiments, the illustrative immunogenic portions may include peptides in which the N-terminal leader sequence and / or the transmembrane domain has been deleted. Other illustrative immunogenic portions will contain a small N- and / or C-terminal deletion (e.g., amino acids 1-30, especially amino acids 1-15) relative to the mature protein.

In another embodiment, the polypeptide composition may comprise one or more polypeptides that are immunologically reactive with T cells and / or antibodies raised against the polypeptide, particularly a polypeptide that has an amino acid sequence or an immunogenic fragment or variant thereof disclosed herein.

In another embodiment, polypeptides are provided that are composed of one or more polypeptides that are capable of inducing T cells and / or antibodies that are immunologically reactive with one or more of the polypeptides described herein, or one or more polypeptides encoded by contiguous nucleic acid sequences contained in the polynucleotides disclosed herein, or immunogenic fragments or variants thereof.

Polypeptides at the N-terminal end of a protein may contain a signal (or leader) sequence which directs transport of the protein, either simultaneously with or after translation. To facilitate synthesis, purification, or identification of a polypeptide (e.g. poly-His), it may also be conjugated to a linker or other sequence, or the binding of the polypeptide to a solid support may be enhanced. For example, the polypeptide can be conjugated to the Fc region of an immunoglobulin.

Among other illustrative embodiments, the polypeptide may be a fusion polypeptide that includes multiple polypeptides as described herein, or that includes at least one polypeptide as described herein and an unrelated sequence such as a known tumor protein. For example, the fusion partner may participate in the provision of T helper epitopes (immunological fusion partner), preferably T helper epitopes recognized by humans, or may participate in the expression of the protein (expression enhancer) with an efficiency greater than that of the native recombinant protein. Some preferred fusion partners are both immunological and expression enhancing partners. Other fusion partners may be selected in such a way as to increase the solubility of the polypeptide or to allow the polypeptide to reach desired intracellular compartments. Still further fusion partners include affinity tags that facilitate the purification of the polypeptide.

Fusion polypeptides can generally be prepared using standard techniques, including chemical conjugation. The fusion polypeptide is preferably expressed as a recombinant polypeptide, allowing the expression system to produce higher levels of the fusion polypeptide relative to the unfused polypeptide. Briefly, DNA sequences encoding polypeptide components can be collected separately and ligated into an appropriate expression vector. The 3 'end of the DNA sequence encoding one polypeptide component is ligated with or without a peptide linker, to the 5' end of the DNA sequence encoding the second polypeptide component such that the reading frames of the sequence are in phase. This allows translation into a single fusion polypeptide that retains the biological activity of both constituent polypeptides.

The peptide linker sequence can be used to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide assembles into its secondary and tertiary structure. Such a peptide linker sequence is inserted into the fusion polypeptide using standard techniques well known in the art. Appropriate peptide linker sequences can be selected based on the following factors: (1) their ability to adapt to a flexible extended conformation, (2) their inability to adapt to a secondary structure that would interact with functional epitopes on the first and second polypeptides, (3) lack of hydrophobic or charged residues that would react with functional antigenic determinants of the polypeptides. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other nearly neutral amino acids such as Thr and Ala can also be used in the linker sequence. Amino acid sequences that can usefully be used as linkers include those described by Marate et al., Gene 40: 39-46, 1985, Murphy et al., Proc. Natl. Acad. Sci USA 83: 8258-8262, in US Patent Nos. 4,935,233 and US 4,751,180. The linker sequence can generally be from 1 to about 50 amino acids in length. Linker sequences are not required where the first and second polypeptides lack essential N-terminal amino acid regions that can be used to separate functional domains and prevent spherical disturbance.

The ligated DNA sequences are operably linked to appropriate transcriptional or translational regulatory elements. Regulatory elements responsible for expression of DNA are located only 5 'to the DNA sequence encoding the first polypeptides. Similarly, the stop codons required for termination of translation and transcription termination signals are present only 3 'to the DNA sequence encoding the second polypeptide.

As described, a fusion polypeptide may contain the polypeptide together with a non-related immunogenic protein, such as an immunogenic protein capable of displaying a boost reaction. Examples of such proteins include tetanus, tuberculosis, and hepatitis proteins (see, for example, Stoute et al., New Engl. J. Med., 336: 86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derived from Mycobacterium sp., Such as an Ra12 fragment derived from Mycobacterium tuberculosis. Ra12 compositions and methods

Their use in increasing the expression and / or immunogenicity of heterologous polynucleotide / polypeptide sequences is known from US Patent Application No. 60/158585. Briefly, Ra12 refers to a polynucleotide region that is a sequence of the MTB32A nucleic acid from Mycobacterium tuberculosis. MTB32A is a 32 KD serine protease encoded by the gene in virulent and avirulent strains of M. tuberculosis. The nucleotide and amino acid sequence of MTB32A has been described (see, for example, US Patent Application No. 60/158585, see also Skeiky et al., Infection and Immun. (1999) 67: 39984007). The C-terminal fragments of the MTB32A coding sequence are expressed at high levels and remain as soluble polypeptides during the purification process. Moreover, Ra12 can increase the immunogenicity of heterologous immunogenic polypeptides with which it has been fused. One preferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotides generally contain at least about 15 contiguous nucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides which encode a portion of the Ra12 polypeptide. The Ra12 polypeptides may contain the native sequence (that is, an endogenous sequence that encodes an Ra12 polypeptide, or a portion thereof), or may contain a variant of such sequence. The Ra12 polynucleotide variant may contain one or more substitutions, additions, deletions, and / or insertions such that the biological activity of the encoded fusion polypeptide is not substantially reduced compared to a fusion polypeptide comprising the natural Ra12 polypeptide. Variants preferably have at least about 70% identity, preferably at least about 80% identity, and more preferably at least about 90% identity to the polynucleotide sequence which encodes the natural Ra12 polypeptide or a portion thereof.

In other preferred embodiments, the immune fusion partner is derived from protein D, a gram negative surface protein from Haemophilus influenza B (WO 91/18926). The protein D derivative preferably comprises approximately one third of the protein (e.g. the first 100-110 N-terminal amino acids) and the protein D derivative may be lipidized. In some preferred embodiments, the first 109 residues of the fusion partner, lipoprotein D, are located N-terminally providing the polypeptide with additional exogenous T cell antigenic determinants and increasing the level of expression in E. coli (thus functioning as an expression enhancer). The lipid terminal part ensures optimal antigen presentation to the antigen presenting cells. Other fusion partners include the non-structural protein from the influenza virus, NS1 (haemagglutinin). Typically 81 N-terminal amino acids are used, although various fragments can be used which contain antigenic determinants that support T.

In another embodiment, the immunological fusion partner is a protein known as LYTA or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae which synthesizes N-acetyl L-alanine amidase known as LYTA amidase (encoded by the LytA gene, Gene 43: 265-292, 1986). LYTA is an autolysin that specifically breaks down some bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity for choline or some choline analogs such as DEAE. This property was used to develop E. coli C-LYTA expressing plasmids useful for the expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10: 795-798, 1992). In a preferred embodiment, the repeating portion of LYTA may be incorporated into the fusion polypeptide. A repeat portion is located in the C-terminal region beginning at residue 178. A particularly preferred repeating portion is comprised of residues 188-305.

Yet another illustrative embodiment includes fusion polypeptides and polynucleotides encoding them, wherein the fusion partner contains a target signal capable of targeting the polypeptide to an endosomal / lysosomal compartment of the cell, as described in US Patent No. 5,633,234. The immunogenic polypeptide, when fused to this target signal, will bind more efficiently to class II MHC molecules and thus provide greater in vivo stimulation of polypeptide-specific ^{CD4 + T cells.}

The polypeptides described herein are obtained using any of the well known techniques of synthesis and / or recombination. Polypeptides, portions, and variants thereof, which are generally less than about 150 amino acids, can be synthesized using techniques well known to those skilled in the art. In one illustrative example, such polypeptides are synthesized using any of the available solid phase techniques such as the Merrifield solid phase method in which amino acids are sequentially added to a growing amino acid chain (see Merrifield, J. Am. Chem. Soc. 85: 2149-2146,1963). Equipment for automatic polypeptide synthesis is commercially available from suppliers such as Perkin Elmer / Applied Bio-Systems Division (Foster City, CA) and may be handled according to the manufacturer's instructions.

The polypeptide compositions (containing the fusion polypeptides) are isolated, and the "secreted" polypeptide is a polypeptide that is removed from its initial environment. For example, a naturally occurring protein or polypeptide is secreted when it is separated from some or all of the coexisting materials in the natural system. Such polypeptides are also preferably purified and have a purity of at least about 90%, especially at least about 95%, and more particularly at least about 99%.

In other aspects of the present disclosure, compounds are provided that contain one or more polynucleotides that encode a polypeptide antigen as set forth above. The terms "DNA" and "polynucleotide" are used herein essentially interchangeably to refer to a DNA molecule that has been released free from all genomic DNA of a particular species. As used herein, the term "isolated" means that the polynucleotide is substantially separate from other coding sequences and that the DNA molecule does not contain large amounts of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this applies to the originally separated DNA molecule and does not exclude genes or coding regions later added to the segment by human hand.

The polynucleotides may contain a native sequence (i.e., an endogenous sequence that encodes a polypeptide / protein of the disclosure, or a portion thereof) or may contain a sequence that encodes a variant or derivative, particularly an immunogenic variant or derivative of such sequence. Variant polynucleotides will typically contain one or more substitutions, additions, deletions, and / or insertions, preferably such that the immunogenicity of the polypeptide encoded by the polynucleotide variant is substantially not less than that of the polypeptide encoded by the polynucleotide sequence provided herein. It is also understood that the term "variants" includes homologous genes of xenogeneic origin.

As noted above, in some preferred embodiments, the polynucleotides described above, e.g., polynucleotide variants, fragments, and hybridizing sequences, encode polypeptides that are immunologically reactive with an antigenic or immunogenic polypeptide. In other preferred embodiments, such polynucleotides encode polypeptides that have a level of immunogenic activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of the activity of the polypeptide sequence specifically set out above.

The polynucleotides described herein, or fragments thereof, regardless of the length of the coding sequence itself, can be combined with other DNA sequences such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other encoding segments, etc. length can vary considerably. Hence, it is contemplated that a nucleic acid fragment of almost any length can be used, with the overall length advantageously being limited by the ease of preparation and use in the recombinant DNA protocol envisaged. For example, in many embodiments, illustrative polynucleotide segments having overall lengths of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs, etc. (including all intermediate lengths).

Compositions can be identified, prepared and / or processed using any of a number of well known techniques (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989, and other references). For example, a polynucleotide can be identified as described in more detail below by screening a DNA array for tumor-associated expression (i.e., an expression that is at least twice as high in a tumor as it is in normal tissue, as determined using a representative assay provided herein). Such searches can be performed, for example, using microarray technology from Affymetrix, Inc. (Santa Clara, CA) according to the manufacturer's instructions (and essentially as described by Schen et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996, and Heller et al., Proc. Natl. Acad. Sci. USA 94: 2150-2155,1997). Alternatively, the polynucleotides may be amplified with cDNA obtained from cells expressing the proteins described above, such as tumor cells.

PL 220 536 B1

A variety of template methods are available for amplifying a target sequence of interest present in a sample. One of the best known amplification methods is the Polymerase Chain Reaction (PCR ™), which is known in more detail from US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159. Briefly, PCR ™ produces two primer sequences that are complementary to regions on opposite complementary strands of the target sequence. To the reaction mixture, excess deoxynucleoside triphosphates are added together with the DNA polymerase (e.g., Taq polymerase). If the target sequence is included in the sample, the primers will bind to the target and the polymerase will cause the primers to extend along the target sequence by addition on the nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target sequence to form reaction products, and excess primers will bind to the target sequence and to the reaction product, and the process is repeated. The reverse transcription and PCR ™ amplification procedure can advantageously be performed to quantify the amount of amplified mRNA. Polymerase chain reaction methodologies are well known in the art.

A variety of other template-dependent methods are well known in the art and are available, many of which are variations on the PCR ™ amplification technique. By way of illustration, some such methods include a ligase chain reaction (referred to as LCR), such as from European Patent Application No. 320308 and U.S. Patent No. 4,883,750, Qbeta Replicase, described in International Patent Application No. PCT / US87 / 00880, strand displacement amplification (SDA Repair Chain Reaction RCR. Still other amplification methods are known from British Patent Specification No. 2202328 and International Patent Application No. PCT / US89 / 01025. Other approaches to nucleic acid amplification include transcription based amplification (TAS) systems (PCT International Patent Application No. WO 88/10315), including nucleic acid sequence based amplification (NASBA) and 3SR. European Patent Application No. 329822 discloses a nucleic acid amplification method which consists in the cyclic synthesis of single-stranded RNA ("ssRNA"), ssDNA and double-stranded DNA (dsDNA). PCT International Patent Application No. WO 89/06700 discloses a nucleic acid sequence amplification scheme based on the hybridization of a promoter / primer sequence to a single-stranded DNA ("ssDNA") target followed by the transcription of multiple copies of the RNA sequence. Other amplification methods, such as "RACE" (Frohman, 1990) and "unilateral PCR" (Ohara, 1989), are also very familiar with those skilled in the art.

An amplified portion of a polynucleotide can be used to isolate a full-length gene from an appropriate library (e.g., a cancer cDNA library) using well-known techniques. In such techniques, the library (cDNA or genomic) is screened using one or more probes or polynucleotide primers suitable for amplification. The library is preferably sized to include larger molecules. Major random libraries may also prove beneficial in identifying 5 'and further gene regions. Genomic libraries are advantageous for making introns and extending 5 'sequences.

For hybridization techniques, a partial sequence may be labeled (e.g., by translation or end-labeling of a cross with ³² P) using well known techniques. The bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or bands containing phage plates) to a labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY. , 1989). Colonies or hybridizing plates are selected and spread out, and DNA is isolated for further analysis. CDNA clones can be analyzed to determine the amount of additional sequence, for example by PCR using a primer from a partial sequence and a primer from a vector. Restriction maps and partial sequences can be generated to identify one or more overlapping clones. The complete sequence can then be determined using standard techniques which may involve the production of a series of deleted clones. The resulting overlapping sequences can then be assembled into a single contiguous sequence. A full-length cDNA molecule can be produced by ligating appropriate fragments using well-known techniques.

Alternatively, amplification techniques, such as the techniques described above, may prove useful in obtaining a full-length coding sequence from a partial cDNA sequence. One such amplification technique is reverse PCR (see Triglia et al., Nucl. Acids Res. 16: 8186, 1988), which uses restriction enzymes to generate a fragment in a known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template

PL 220 536 B1 for PCR with divergent primers from a known region. In an alternative approach, sequences adjacent to a partial sequence can be recovered by primer amplification for a linker sequence and a known region-specific primer. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second region-specific primer. A variant of this procedure, which uses two primers which initiate expansion in opposite directions from a known sequence, is known from WO 96/38591. Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique relies on the use of an inner primer and an outer primer that hybridizes to the polyA region or vector sequence to identify sequences that are 5 'and 3' of a known sequence. Additional techniques include capture PCR (Langerstrom et al., PCR Methods Applic. 1: 111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res. 19: 3055-60, 1991). Other methods that use amplification can also be used to obtain full-length cDNA sequences.

In some circumstances, it is possible to obtain full-length cDNA sequences by sequence analysis performed on an expressed sequence tag (EST) database, such as is available from the GenBank gene bank. Generally, the search for overlapping ESTs may be performed using well-known programs (e.g., NCBI BLAST searches) and such ESTs may be used to generate full-length contiguous sequences. Full-length DNA sequences can also be obtained by genomic fragment analysis.

In other embodiments, polynucleotide sequences, or fragments thereof, that encode the polypeptides set forth above, or fusion proteins, or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of the polypeptide in appropriate host cells. As a result of the spontaneous degeneracy of the genetic code, other DNA sequences can be obtained that encode substantially the same or a functionally equivalent amino acid sequence, and these sequences can be used for cloning and expression of a given polypeptide.

Sequences encoding a desired polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, MH et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. ( 1980) Nucl. Acids Res. Symp. Ser. 225-232).

To express the desired polypeptide, the nucleotide sequences encoding the polypeptide, or functional equivalents thereof, can be inserted into an appropriate expression vector, i.e., a vector that contains the elements necessary for transcription and translation of the inserted coding sequence. Methods well known to a person skilled in the art can be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. These techniques are described, for example, by Sambrook, J. et al. (1989) in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.M. et al (1989) in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

The "control elements" or "regulatory sequences" contained in the expression vector are those untranslated regions of the vector - enhancers, promoters, 5 'and 3' untranslated regions - that interact with host cell proteins to effect transcription and translation. Such elements may vary in strength and specificity. Depending on the vector and host system used, any number of appropriate transcriptional and translational elements may be used, including constitutive inducible promoters.

A number of virus-based expression systems are generally available in mammalian cells, and, for example, in cases where an adenovirus is used as the expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription / translational complex consisting of the late promoter and tripartite sequence. leading. Insertion in the irrelevant E1 or E3 region of the viral genome can be used to obtain a virulent virus that can express the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655- 3659). In addition, transcription enhancers such as Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells.

Specific initiation signals can also be used to achieve more efficient translation of the coding sequences of the polypeptide of interest. Such signals include the initiating ATG codon

PL 220 536 B1 and adjacent sequences. In those cases where the polypeptide coding sequences, its initiation codon, and the sequences that follow are inserted into an appropriate expression vector, no additional transcriptional and translational control signals may be required. However, in those cases where only or part of a coding sequence is introduced, the exogenous translational control signals including the ATG initiation codon should be anticipated. Furthermore, in order to ensure translation of the entire insertion, the initiation codon should be in the correct reading frame. Exogenous translation elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be increased by inclusion of enhancers that are appropriate for the particular cell system used, such as that described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162).

A number of protocols for detecting and measuring the expression of polynucleotide-encoded products using product-specific polyclonal or monoclonal antibodies are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sort (FACS). In some applications, it may be advantageous to use a monoclonal two-site immunoassay using monoclonal antibodies reactive with non-interfering antigenic determinants on a given polypeptide, and a competitive binding assay may also be used. These and other trials are described, inter alia, by Hampton R. et al. (1990, Serological Methods, a Laboratory Manual, APS Press, St. Paul. Minn.) And Maddox, D.E. et al (1983, J. Exp. Med. 158: 1211-1216).

Pharmaceutical Compositions and Methods

It is understood that, if desired, the compounds disclosed herein may be administered in combination with other therapeutic modalities, such as antibacterial, antiviral, and antifungal compounds, or therapies, various DNA-based therapeutics, RNA-based therapeutics, on polypeptides and / or with other immunoeffectors. In fact, in principle, any other component may be included as long as the additional component (s) does not have a significant adverse effect on contact with the target cells or host tissues. The compositions may therefore be introduced together with various other agents as required or desired for the specific embodiment (s) of the invention being implemented. By way of illustration, pharmaceutical compositions may contain or may be used in conjunction with DNA encoding one or more therapeutic proteins, antisense RNAs, ribosomes, etc.

In one aspect, the compounds of the invention and compositions containing them may be administered together with an antigen to provide an adjuvant or enhancement effect to the antigen, that is, to enhance the immune response of a patient or subject. In another aspect, the compounds of the invention and compositions containing them are administered, in the absence of any exogenous antigen, for the therapeutic effect of the compound itself.

In another aspect where the compound or composition is administered together with an exogenous antigen, methods have been developed for the treatment, amelioration and / or significant prevention of infectious diseases in eukaryotic patients, particularly animals, and particularly humans. Given the importance of TLR-dependent signaling in the innate immune response to microbial challenge, the ability to stimulate such pathways selectively and with minimal toxicity represents a powerful agent for prophylactic and / or therapeutic modalities of treatment against a wide range of infectious agents.

The methods described herein are applicable to virtually any type of infectious agent, including bacteria, viruses, parasites, and fungi. By way of illustration, the invention is useful for the prophylactic and / or therapeutic treatment of bacterial infections by strains from Pseudomonas, Escherichia, Klebsiella, Enterobacter, Proteus, Serratia, Candida, Staphylococci, Streptccocci, Chlamydia, Mycoplasma and many others. Illustrative viral disease conditions that can be treated using the compounds of the invention include those caused, for example, by influenza viruses, adenoviruses, parainfluenza viruses, rhinitis viruses, respiratory viruses (RSV), herpes viruses, cytomegaloviruses, hepatitis viruses, for example hepatitis B and C viruses, and others. Illustrative fungi include, for example, Aspergillis, Candida albicans, Cryptococcus neoformans, Coccidioides immitus, and others.

In one illustrative embodiment, methods of treating patients, especially immunocompromised patients who have developed, or are at risk of developing, are described herein.

Infections such as nosocomial bacterial and viral infections. About 2 million of the 40 million hospitalized patients develop infection each year during their hospital stay, and about 1% or about 400,000 patients develop nosocomial pneumonia and more than 7,000 die. This makes nosocomial pneumonia the leading cause of death from a hospital acquired infection. Thus, in this solution an important need for an effective preventive approach in the treatment of nosocomial infections is met.

In a similar embodiment, prophylactic treatment of immunocompromised patients, such as HIV-positive patients, who have developed or are at risk of developing pneumonia, or from an opportunistic infection, or from reactivation of a suppressed or latent infection, is described herein. In 1992, in the United States of America alone, there were approximately 20,000 cases of Pneumocystis carinii infections in AIDS patients, and an additional 60-70% of all AIDS patients acquire P. carinii after some time of their illness. Thus, effective preventive methods for a population at risk are described herein.

In another similar embodiment, the methods described herein can be used to treat other patient populations who may be immunocompromised or at risk of developing infectious diseases, for example, including patients with cystic fibrosis, chronic obstructive pulmonary disease, and other patients who are immunocompromised. immunity and / or in-house-treated patients.

In another aspect, the compounds of the invention and compositions described herein can be used (in the absence of an exogenous antigen) in methods of treating, ameliorating or significantly preventing allergic disorders and conditions such as sinusitis, chronic rhinitis, asthma, and atopic dermatitis. and psoriasis. Such an approach relies at least in part on the ability of the compounds to activate cytokine production from target cells that can compete with stereotyped allergy-type cytokine responses characterized by IL-4 production or over-reactivity to IL-4 activity. Administration of some of the monosaccharide and disaccharide compounds described herein results in the expression of IFNgamma and IL-12 from antigen processing and containing cells, as well as from other cells that result in decreased cytokine activity associated with allergic reactions, such as IL-4, 5, 6. , 10 and 13.

In yet another aspect, the compounds of the invention and compositions containing them (without exogenous antigen) can be used in methods of treating autoimmune diseases and conditions. The compounds will typically be selected from compounds capable of antagonizing, inhibiting or otherwise negatively modulating one or more Toll receptors, in particular Tlr2 and / or Tlr4, such that the immune response associated with the disease state is improved or significantly prevented. Illustratively, the methods described herein can be used to treat disease states such as inflammatory bowel disease, rheumatoid arthritis, chronic arthritis, multiple sclerosis, and psoriasis.

The compounds of the present invention can also be used as adjuvants and immunoeffectors that increase antibody production in immunized animals, stimulate cytokine production, and stimulate a cell mediated immune response, including the cytotoxic T cell response.

In the methods described herein, for example, for eliciting an immune response in a patient, the compounds of the present invention and compositions containing them can be formulated with a pharmaceutically acceptable injection or ingestion vehicle. The term "pharmaceutically acceptable carrier" as used herein means a medium which does not interfere with the immunomodulatory activity of the active ingredient and is not toxic to the patient to whom it is administered. Pharmaceutically acceptable carriers include oil-in-water and water-in-oil emulsions, aqueous compositions, liposomes, microbeads, and microsomes. For example, the carrier can be a microsphere or microparticle that contains a compound of the present invention within the matrix of the sphere or particle, or adsorbed on the surface of the sphere or particle. The carrier can also be an aqueous solution or micellar dispersion containing triethylamine, triethanolamine or other agent which makes the composition alkaline, or a suspension containing aluminum hydroxide, calcium hydroxide, calcium phosphate or tyrosine adsorbate. The carriers may also contain other solvents, dispersion media, excipients, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. in the field. From lec20

Except where any conventional media or agent is incompatible with the active ingredient, their use in the therapeutic compositions is contemplated.

Compositions of the compounds of the present invention which may be administered parenterally, i.e. intraperitoneally, subcutaneously or intramuscularly, contain the following preferred carriers. Examples of preferred carriers for subcutaneous use include phosphate buffered saline (PBS) and 0.01-0.1% triethanolamine in USP water for injection. Suitable carriers for intramuscular injection include 10% USP ethanol, 40% propylene glycol, and balance in an acceptable isotonic solution such as 5% dextrose.

Examples of preferred intravenous carriers include 10% USP ethanol, 40% USP propylene glycol, and USP water balance for injection. Another acceptable carrier comprises 10% USP ethanol and USP water for injection, and yet another acceptable carrier comprises 0.01-0.1% triethanolamine in USP water for injection. Pharmaceutically acceptable parenteral solvents are those solvents which give a solution or dispersion which can be filtered through a 5 micron filter without removing the active ingredient.

The preferred method of administering the compositions described herein is mucosal administration, particularly intranasal administration, or administration by inhalation (pulmonary administration). Drug delivery to the lungs can be achieved by several different approaches, including liquid nebulizers, aerosol-based metered dose inhalers (MDIs), and dry powder dispersing devices. Compositions for use in this type of administration are typically dry powders or sprays. When administering aerosols, which are the recommended mode of administration, the compositions are delivered by means of inhalers, some types of which are described below.

Dry powders contain, in addition to the active ingredient, a carrier, an absorption-enhancing agent, and optionally other ingredients. The carrier is, for example, a mono-, di- or polysaccharide, sugar alcohol or other polyol. Suitable carriers include lactose, glucose, raffinose, melesitose, lactitol, maltitol, trehalose, sucrose, mannitol and starch, with lactose being especially preferred, especially in the form of its monohydrate. Carriers also include absorption enhancers such as polypeptides, surfactants, alkylglycosides, amino fatty acid salts, or phospholipids. The components of the compositions typically must be in a particulate form, i.e., their volume median diameter should generally be from about 30 to about 200 microns as measured by a laser diffraction instrument or Coulter counter. The desired particle size can be obtained by methods known in the art, for example by milling, micronization or direct precipitation.

The nasal route of administration provides numerous advantages over other routes of administration of the compounds of the present invention. One of the advantages of nasal administration is convenience. The injection system requires sterilization of the hypodermic syringe and the environment of the establishment, leading to care among medical personnel about the risk of acquiring disease by accidental pricking with a contaminated needle. Strict requirements for the safe disposal of used needles and syringes must also be imposed in the plant environment. In contrast, intranasal administration requires little time on the part of the patient and the medical personnel involved and is far less burdensome for the establishment than for injections.

A second important advantage of nasal administration is patient acceptance of the drug delivery system. Nasal administration is perceived as non-invasive, with no pain, no significant follow-up, and the satisfaction of rapidly resolving the discomfort in a symptomatic patient. This is especially beneficial when the patient is a child. Another important consideration is that the patient may self administer the prescribed dose (s) of the nasal spray.

For nasal administration, the compositions described herein may be formulated as liquids or solids. Such compositions may contain one or more adjuvants, agents to enhance the absorption of the active ingredients by passage through the nasal membrane and (in the case of liquid compositions) an aqueous diluent, for example water. Alternatively, the diluent may contain an aqueous buffer such as a phosphate buffer. The composition may further optionally contain one or more polyols and one or more preservatives, such as, for example, gentamicin, bacitracin (0.005%) or cresol. The compositions can be administered into the nasal cavity in the form of a spray using an atomizer, nebulizer, nebulizer, dropper, or other device which brings the solution into contact with the nasal mucosa. The device may be a simple device such as a simple nasal spray that may be used by a patient

Or may be a more complex device for more accurate dosing of the composition, which may be used in a doctor's office or medical facility.

Nasal powder compositions can be prepared by mixing the active ingredient and the excipient, both having the desired particle size. First, a solution of the active ingredient and cyclodextrin excipients is prepared, followed by precipitation, filtration, and powdering. It is also possible to remove the solvent by lyophilization and then pulverization of the powder to the desired particle size using conventional techniques known from the pharmaceutical literature. A final step is size grading, for example by sieving, to obtain particles which preferably have a diameter of 30 to 200 microns. The powders can be administered using a nasal insufflator, or they can be packaged in an inhalation or insufflation device. The needle penetrates the bag creating pores at the top and bottom of the bag and air is introduced to blow the powder particles away. The powder composition can also be administered under an inert gas stream or suspended in liquid organic liquids.

In a specific embodiment, the pharmaceutical composition may be delivered in a controlled release or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see Langer, Science, 249: 15271533 (1990), Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 10, Buschwald et al. 1980, Surgery 88: 507, Saudek et al. 1989 N. Engl. J. Med. 321: 574). Alternatively, polymeric materials can be used to achieve controlled or sustained release of a κ-opioid agonist and / or an opioid antagonist (see, for example, Medical Applications of Controlled Release, Langer and Wise (editors), CRC Pres., Boca Raton, Florida 1974) , Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (editors), Wiley, New York (1984), Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macrol. Chem. 23: 61, see also Levy et al., 1985, Science 228: 190, During et al., 1989, Ann. Neurol. 25: 351, Howard et al., 1989, J. Neurosurg. 71: 105, U.S. Patent Nos. 5,679,377, 5916597, 5912015, 5989463, 5128326, PCT Application No. WO 99/12154 and PCT Application No. WO 99/20253). Examples of polymers used in sustained-release compositions include, but are not limited to, polymethyl methacrylate, polymethyl methacrylate, polyacrylic acid, polyethylene vinyl acetate, poly methacrylic acid, polyglycolides (PLG), polyanhydrides, poly-N-vinylpyrrolidone, polyvinyl alcohol, polyvinyl alcohol ethylene, polylactides (PLA), polylactoglycolides (PLGA) and polyorthoesters. In a preferred embodiment, the polymer used in the sustained release composition is inert, free of leachable contaminants, storage-stable, sterile, and biodegradable. In yet another embodiment, the controlled-release or sustained-release system can be placed close to the therapeutic target, thus requiring only a fraction of the systematic dose (see, for example, Goodson in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). .

Carriers for use with such pharmaceutical compositions are biocompatible carriers and can also be biodegradable. In some embodiments, the composition provides a relatively constant level of active ingredient release. In other embodiments, however, a faster release rate immediately after administration may be desirable. The composition of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of polylactoglycolide, polyacrylate, latex, starch, cellulose, dextran, etc. Other illustrative delayed release carriers include supramolecular biovectors that have a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and an optional outer layer. an amphiphilic compound such as a phospholipid (see, for example, US Patent No. 5,151,254 and PCT Patent Application Nos. WO 94/20078, WO / 94/23701, and WO 96/06638). The amount of active compound contained in the sustained release composition depends on the site of implantation, the rate and duration of release expected, and the nature of the disease state or condition being prevented.

The compounds of the present invention can be administered to a patient in an effective amount or a pharmaceutically effective amount to achieve or enhance the immune response in the patient. The term "effective amount" or "pharmaceutically effective amount" as used herein is that amount that responds superior to the vehicle or negative controls. An "adjuvant-effective amount" is that amount of a given compound which, when administered in combination with an antigen, shows a greater response than that produced by the antigen alone. Exact dosage of we22 compounds

The administration of the subject invention to a patient will depend on the particular compound employed, the route of administration, the pharmaceutical composition, and the patient. For example, when administered subcutaneously to enhance antibody response, the amount of the compound used is from 1 to about 250 micrograms, and more preferably from about 25 to about 50 micrograms, based on a typical 70 kg adult patient.

In another embodiment, illustrative immunogenic compositions, for example, immunogenic compositions and / or vaccines, contain DNA encoding one or more polypeptides as described above such that the polypeptide is generated in situ. As noted above, the polynucleotide may be administered in any of a variety of delivery systems known to one of ordinary skill in the art. In fact, many gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, 1998, and references cited therein. Suitable polynucleotide expression systems will, of course, include the necessary regulatory DNA sequences for expression in the patient (such as a suitable promoter and termination signal).

Thus, in some embodiments, the polynucleotides encoding immunogenic polypeptides described herein are introduced into appropriate mammalian host cells for expression using a variety of known virus-based systems. In one illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. The selected nucleotide sequence encoding the polypeptide can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be secreted and delivered to the patient. A number of illustrative retroviral systems have been described (see, for example, U.S. Patent No. 5,219,740, Miller and Rosman (1989) BioTechniques 7: 980-990, Miller, AD (1990) Human Gene Therapy 1: 5-14, Scarpa et al. (1991) Virology 180: 849-852, Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037, and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: 102-109 .

In addition, a number of illustrative adenovirus based systems have also been described. Unlike retroviruses that integrate into the host genome, adenoviruses persist beyond the chromosomes and thus minimize the risks associated with insertion-related mutagenesis (HajAhmad and Graham (1986) J. Virol. 57: 267-274, Bett et al. ( 1993) J. Virol. 67: 5911-5921, Mittereder et al. (1994) Human Gene Therapy 5: 717-729, Seth et al. (1994) J. Virol. 68: 933-940, Barr et al. (1994) Gene Therapy 1: 51-58, Berkner, KL (1988) BioTechnics 6: 616-629, and Rich et al. (1993) Human Gene Therapy 4: 461-476).

Various vector systems based on adeno-associated virus (AAV) have also been developed for delivery of polynucleotides. AAV vectors can be readily constructed using techniques well known in the art (see, for example, US Patent Nos. 5,173,414 and 5,139,941, PCT International Patent Application Nos. WO 92/01070 and WO 93/03769, Lebkowski et al. (1988) Molec. Cell. Biol. 8: 3988-3996, Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press), Carter, BJ (1992) Current Opinion in Biotechnology 3: 533-539, Muzyczka, N. (1992) Current research in Microbiol. And Immunol. 158: 97-129, Kotin, RM (1994) Human Gene Therapy 5: 793-801, Sheiling and Smith (1994) Gene Therapy 1: 165-169, and Zhou et al. (1994) J. Exp Med. 179: 1867-1875.

Additional viral vectors useful for gene transfer of polynucleotides encoding the polypeptides described herein include viral vectors derived from the pox family of viruses such as vaccinia virus and avipox virus. By way of example, a recombinant vaccinia virus expressing new molecules can be constructed as follows. The DNA encoding the polypeptide is first inserted into a suitable vector such that it is adjacent to the vaccinia promoter and flanking vaccinia DNA sequences, such as the thymidine kinase (TK) encoding sequence. This vector is then used to transfect cells that are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The obtained recombinant sup (-) TK can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and selecting resistant viral plaques.

The vaccinia-based infection / transfection system may conveniently be used to provide inducible, transient expression or co-expression of one or more of the polypeptides described herein in host cells of the organism. In this particular system, cells are first infected in vitro with recombinant vaccinia virus that encodes bacteriophage T7 RNA polymerase. This polymerase exhibits an excellent specificity in that it is only transcribed

Of matrices containing T7 promoters. Following infection, cells are transfected with the polynucleotide or polynucleotides of interest using the T7 promoter. Cytoplasmic polymerase from recombinant vaccinia virus transcribes the transfected DNA into RNA, which is then translated into the polypeptide through the host's translational machinery. Such a method provides for transient, high-level cytoplasmic production of large amounts of RNA and its translation products (see, for example, Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87: 6743-6747, Fuerst et al., Proc Natl Acad Sci USA (1986) 83: 8122-8126.

Alternatively, avipox viruses, such as fowlpox and canarypox viruses, can also be used to introduce coding sequences of interest. Recombinant avipox viruses expressing immunogens from mammalian pathogens are known to confer protective immunity when administered to non-avian species. The use of the Avipox vector is particularly desirable in humans and other mammalian species, because members of the genus Avipox can replicate efficiently in the corresponding avian species and therefore do not succumb to infection in mammalian cells. Methods for producing recombinant avipox viruses are known in the art and employ genetic recombination as described above for the production of pox viruses (see for example WO 91/12882, WO 89/03429 and WO 92/03545).

A variety of alphavirus vectors can also be used to deliver the polynucleotide compositions described herein, such as those known from US Patent Nos. 5,843,723, 6,015,686, 6,008,035, and 6,015,694. Certain Venezuelan equine encephalitis (VEE) vectors may also be used, illustrative examples of which can be found in US Patent Nos. 5,505,947 and 5,645,576.

In addition, molecular coupled vectors can also be used for gene delivery, such as the adenoviral chimeric vectors described by Michael et al in J. Biol. Chem. (1993) 268: 6866-6869, and Wagner et al. In Proc. Natl. Acad. Sci. USA (1992) 89: 6099-6103.

Additional illustrative information on these and other known virus-based delivery systems can be found, for example, in Fischer-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321,1989, Flexner et al., Ann. N.Y. Acad. Sci. 569: 86-103, 1989, Flexner et al., Vaccine 8: 17-21, 1990, U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487, WO 89/01973, US 4,777,127, GB 2200651, EP 0345242, WO 91 / 02805, Berkner, Biotechnigues 6: 616-627, Rosenfeld et al., Science 252: 431-434, 1991, Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219,1994, Kass-Esler et al., Proc. Natl. Acad. Sci. USA 90: 11498-11502, 1993, Guzman et al, Circulation 88: 2838-2848, 1993, and Guzman et al. Cir. Res. 73: 12021207,1993.

In some embodiments, the polynucleotide can be integrated into the genome of the target cell. Such integration may take place at a specific site and orientation by homologous recombination (gene replacement) or may be introduced at a random, non-specific site (gene augmentation). In still further embodiments, the polynucleotide may be held stably in the cell as a separate, episomal DNA segment. Such polynucleotide stretches or "episomes" encode sequences sufficient to allow for maintenance and replication independent of, or in synchronization with, the host cell cycle. The manner in which the expression construct is brought into the cell, where the polynucleotide remains in the cell, depends on the type of expression construct used.

In another embodiment, the polynucleotide is administered / delivered in the form of "naked" DNA as described, for example, in Ulmer et al., Science 259: 1745-1749, 1993, and reviewed in Cohen, Science 259: 1691-1692, 1993. DNA can be increased by coating the DNA on biodegradable beads which are efficiently transported into cells.

In yet another embodiment, a composition as described herein can be delivered by particle bombardment, many of which have been described. In one illustrative example, acceleration of the gas driven particles can be accomplished with equipment such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, WI), several examples of which are known from U.S. Patent Nos. 5,846,796, 6,010,478, 5,865,796, 5,584,807, and EP 0500799. Such an approach offers needle-free delivery in which a dry powder composition of microscopic particles such as polynucleotide or polypeptide particles , accelerates to high speed in the helium gas stream generated by the hand-held device, driving the particles into the target tissue of interest.

PL 220 536 B1

In a similar embodiment, other devices and methods that may be useful for the gas-driven, needle-free injection of the compositions described herein include devices and methods provided by Bioject, Inc. (Portland, OR), several examples of which are known from U.S. Patent Nos. 4,790,824, 5,064,413, 5,312,335, 5,383,851, 5,399,163, 5,520,639, and 5,993,412.

In some embodiments, the pharmaceutical composition is preferably a composition that elicits a predominantly Th1 immune response. High levels of Th1-type cytokines (e.g., IFN-γ, IFNa, IL-2, and IL-12) tend to promote the elicitation of immune responses mediated by cells against the administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6, and IL-10) tend to promote the induction of humoral immune responses. Upon use of the immunogenic composition formulated herein, the patient will develop an immune response that includes both Th1 and Th2 responses. In a preferred embodiment, where the response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines can be easily determined using standard tests. Alternatively or additionally, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6, and IL-10) may be desirable for certain therapeutic applications. The levels of these cytokines can be easily determined using standard tests. For a review of the cytokine families, reference is made to Mossmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

Illustrative compositions for use in inducing Th1-type cytokines include, for example, a combination of CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated), as described, for example, in WO 96/02555, WO 99/33488, and US Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example by Sato et al., Science 273: 352, 1996. Other immunostimulatory agents include saponins such as QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA) and similar derivatives of saponins and their mimetics.

Any of a variety of additional immunostimulatory agents, for example, cytokines such as GM-CSF, interferons, or interleukins, may be included in the compositions described herein to further modulate the immune response of interest, in addition to Montanide ISA 720 (Seppic, France) immunostimulants, SAF ( Chiron, California, USA), ISCOMS (CSL), MF-59 (Chiron), SBAS series of adjuvants (e.g. SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium) and Enhanzyn ™ (Corixa, Hamilton , MT). In WO 99 / 52549A1, immunostimulants based on polyoxyethylene ethers are described and may also be used.

The pharmaceutical compositions described herein will also often contain one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostatic agents, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), dissolved agents which render the composition isotonic, hypotonic or slightly hypertonic with the recipient's blood, suspending agents, thickening agents and / or preservatives. Alternatively, compositions described herein may be formulated as a lyophilizate.

The pharmaceutical compositions described herein may be contained in single-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are sealed in such a way as to maintain the sterility and stability of the composition until use. Generally, the compositions can be stored as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, the pharmaceutical composition can be stored dry-frozen only requiring the addition of a sterile liquid carrier immediately prior to use.

The development of appropriate dosing and treatment regimens using the particular compositions described herein in a variety of treatment regimens, including, for example, oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation is well known in the art, some of which are briefly discussed in the following section. general purpose illustration.

For some applications, the pharmaceutical compositions described herein can be delivered to an animal by oral administration. The compositions as such may be formulated with an inert diluent or an assimilable edible carrier, or they may be enclosed in a hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly into the food of the diet.

The active compounds can even be incorporated with excipients and used in the form of swallowed tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,

PL 220 536 B1 and the like (see, for example, Mathiowitz et al., Nature, Mar 27, 1997, 386 (6623): 410-4, Hwang et al., Crit Rev Ther Drug Carrier Sits 1998, 15 (3): 243-84. U.S. Patent Nos. 5,641,515, 5,580,579 and 5,792,451). The tablets, troches, pills, capsules, etc. may also contain any of a variety of additional ingredients, and for example, a binder such as gum tragacanth, acacia, corn starch or gelatin, excipients such as dicalcium phosphate, a disintegrating agent, such as such as cereal starch, potato starch, alginic acid and the like, a lubricant such as magnesium stearate, and a sweetening agent such as sucrose, lactose or saccharin, or a flavoring such as peppermint, wintergreen oil or cherry flavor - fragrant. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be included as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. Of course, any material used in preparing any unit dose should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compounds can be incorporated into a sustained release formulation and composition.

Typically these compositions will contain at least about 0.1% or more of active compound, although the percentage of active ingredient (s) may of course vary and is preferably from about 1 or 2% to about 60 or 70% or more by weight. or the volume of the entire composition. Of course, the amount of active ingredient (s) in any therapeutically effective composition may be formulated so as to obtain the appropriate dosage for each given unit dose of the compound. One skilled in the art of preparing such pharmaceutical compositions will take into account factors such as solubility, bioavailability, biological half-life, route of administration, product life, and other pharmacological considerations, and as such a variety of dosages and treatment regimens may be desirable.

For oral administration, the compositions described herein may alternatively be formulated with one or more excipients in the form of a mouth rinse, toothpaste, buccal tablet, mouth spray, or an orally sublingual formulation. Alternatively, the active ingredient may be incorporated into an oral solution such as a solution containing sodium borate, glycerin, and potassium bicarbonate, or suspended in toothpaste, or added in a therapeutically effective amount to a composition which may contain water, binders, abrasives, flavors, foaming agents, and wetting agents. Alternatively, the compositions may be formulated as a tablet or solution which may be placed under the tongue or otherwise dissolved in the mouth.

In some circumstances, it will be desirable to deliver the pharmaceutical compositions described herein by parenteral, intravenous, intramuscular, and even intraperitoneal administration. Such approaches are well known to those skilled in the art, some of which are described further, for example, in US Pat. Nos. 5,543,158, 5,641,515 and 5,399,363. In some embodiments, solutions of the active compounds in free base or pharmaceutically acceptable salt form can be prepared in water preferably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be obtained in glycerin, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, such preparations will generally contain a preservative to prevent the growth of microorganisms.

Illustrative pharmaceutical forms suitable for injection use include sterile aqueous solutions or suspensions and sterile powders for the preparation of sterile injectable solutions or dispersions (see, for example, US Patent No. 5,466,468). In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerin, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol,

Sorbic acid, thimerosal, and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin in the compositions.

In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered, if necessary, and the liquid diluent should be isotonic with an appropriate saline solution or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Accordingly, in light of the present specification, a sterile aqueous medium that can be used will be known to one skilled in the art. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml subcutaneous infusion fluid or injected at a proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). ). Some adjustment of the dose will be necessary depending on the condition of the treated patient. Moreover, when administered to humans, the formulations will of course preferably meet the sterility, pyrogenicity, and general safety and cleanliness standards required by the FDA Office of Biological Standards.

In another embodiment, the compositions described herein may be formulated as neutral or salt forms. Illustrative pharmaceutically acceptable salts include acid addition salts (formed with the amino acid groups of the protein) that are formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or with organic acids such as acetic, oxalic, tartaric, mandelic, etc. Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. Once formulated, the solutions will be administered in in a manner compatible with the composition of the dose, and in such amount as is therapeutically effective.

In some embodiments, the pharmaceutical compositions can be administered by nasal sprays, inhalation, and / or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays are known, for example, from US Patent Nos. 5,756,353 and 5,804,212. Similarly, drug delivery using intranasal microparticulate resins (Takenaga et al., J Controlled Release, Mar 2, 1998, 52 (1-2): 81-7) and lysophosphatidylglycerol compounds (US Patent No. 5,725,871) are well known in the pharmaceutical art. . Similarly, illustrative delivery of drugs through a mucosa in the form of a matrix on a polytetrafluoroethylene backing is known from US Pat. No. 5,780,045.

In some embodiments, liposomes, nanocapsules, lipid particles, vesicles, etc. are used to introduce the compositions described herein into suitable cells / host organism. In particular, the compositions may be formulated for administration either encapsulated in a liquid particle, liposome, vesicle, nanosphere, or nanoparticle. etc. Alternatively, the compositions may be bonded, either covalently or non-covalently, to the surface of such carrier excipients.

The formulation and use of liposomal and liposomal-like formulations as potential drug carriers is generally known to those skilled in the art (see, for example, Lasic, Trends Biotechnol, Jul 1998, 16 (7): 307-21, Takakura, Nippon Rinsho, March 1998, 56 ( 3): 691-5, Chandran et al., Indian J Ex Biol., Aug 1997, 35 (8): 801-9, Margalit, Crit Rev Ther Drug Carrier Syst. 1995, 12 (2-3): 233-61 , U.S. Patent Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other means, including T-cell suspensions, primary hepatic cultures, and PC 12 cells (Renneisen et al., J Biol Chem. Sep 25, 1990, 265 (27) : 16337-42, Muller et al., DNA Cell Biol, Apr. 1990, 9 (3): 231-9). In addition, liposomes are free from DNA length constraints that are typical of viral based delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors, etc., into a variety of cultured cell lines and animals. In addition, the use of liposomes

PL 220 536 B1 does not appear to be related to autoimmune reactions or to unacceptable toxicity when introduced systemically.

In some embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar, concentric bilayer vesicles (also called multilamellar vesicles (MLVs).

Alternatively, in other embodiments, pharmaceutically acceptable nanocapsule formulations of the compositions described herein have been developed. In general, nanocapsules can trap compounds in a stable and reproducible manner (see, for example, Quintanar-Guerrero et al., Drug Dev Ind Pharm., Dec. 1988, 24 (12): 1113-28). To avoid side effects due to intracellular polymer overload, such ultrafine particles (about 0.1 µm in size) can be constructed using polymers that can degrade in vivo. Such particles can be produced as described, for example, by Couvreur et al., Crit Rev Ther Drug Carier Syst. 1988, 5 (1): 1-20, zur Muhlen et al., Eur J Pharm Biopharm., Mar 1998, 45 (2): 149-55, Zambaux et al., J Controlled Reiease, Jan 1998, 50 (1-3 ): 31-40, and in U.S. Patent No. 5,145,684. Cancer therapies

Immunological treatments for cancer are based on the recognition that cancer cells can often escape the body's defenses against abnormal or foreign cells and particles, and that this defense could be stimulated therapeutically to regain lost interest, for example pp. 623-648 in Klein , Immunology (Wiley-Interscience, New York, 1982). The numerous recent observations that various immune effectors can directly or indirectly inhibit tumor growth have led to a renewed interest in this approach to cancer therapy (see, for example, Jager et al., Oncology 2001, 60 (1): 1-7, Renner et al. , Ann Hematol 2000 Dec, 79 (12): 651-9.

The four primary cell types whose function has been implicated in anti-tumor cell immunity and the elimination of cancer cells from the body include i) B lymphocytes, which secrete immunoglobulins into the blood plasma to identify and label non-invasive cells, ii) monocytes that secrete complement proteins that are responsible for the lysis and processing of immunoglobulin-coated, invasive target cells, iii) naturally killing lymphocytes, which have two mechanisms to destroy tumor cells, antibody-dependent cellular cytotoxicity and natural killing, and iv) T cells, which have antigen-specific receptors and demonstrate the ability to recognizing a tumor cell containing complementary marker molecules (Schreiber, H, 1989, in Fundamental Immunology, WE Paul (editor), pp. 923-955).

Cancer immunotherapy generally focuses on eliciting humoral immune responses, cellular immune responses, or both. Moreover, it is generally accepted that the induction of CD4 ⁺ helper cells is necessary in order to secondary induce either antibodies or CD8 ⁺ cytotoxic T cells. Polypeptide antigens that are selective or ideally specific for cancer cells provide a powerful approach in eliciting immune responses against cancer.

The compositions described herein can be used to stimulate immune responses against cancer. In such methods, the pharmaceutical compositions described herein are administered to a patient, typically a warm-blooded animal, and in particular a human. The patient may or may not be affected by cancer. The pharmaceutical compositions and vaccines can be administered either before or after surgical removal of the primary tumors and / or treatment such as the administration of radiotherapeutic or conventional chemotherapeutic drugs. As discussed above, administration of the therapeutic compositions can be by any suitable method, including intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, transdermal, rectal, vaginal, topical and oral administration.

In some embodiments, the immunotherapy can be active immunotherapy, wherein the treatment is based on stimulating in vivo the host's endogenous immune system to respond against tumors with administration of immune response modifying agents (such as polypeptides or polynucleotides as provided herein).

The route and frequency of administration of the therapeutic compositions described herein, as well as the dose, will vary from subject to subject and can be readily determined using standard techniques. In general, pharmaceutical compositions and vaccines can be administered by injection (e.g., intradermal, intramuscular, intravenous, or subcutaneous), intranasally (e.g., aspiration), or orally28.

No. Preferably, from 1 to 10 doses may be administered over a period of 52 weeks. Preferably, 6 doses are administered at 1 month intervals, and booster vaccines may be administered periodically thereafter. Varying protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above baseline (i.e. no treatment). Such a response can be controlled by measuring anti-tumor antibodies in a patient or by vaccine-dependent production of cytolytic effector cells capable of killing the patient's tumor cells in vivo. Such vaccines should also be capable of eliciting an immune response that leads to a better clinical outcome (e.g., more frequent remission, complete or partial or longer disease-free survival) in vaccinated patients as compared to unvaccinated patients. Generally, for pharmaceutical compositions and vaccines containing one or more polypeptides, the amount of each polypeptide included in a dose is from about 25 µg to 5 mg per kg of host weight. Suitable dose sizes will vary with the size of the patient, but will typically be from about 0.1 to about 5 ml.

In general, an appropriate dose and treatment regimen will provide the active compound (s) in an amount sufficient to provide a therapeutic and / or prophylactic effect. Such a response can be controlled by establishing a better clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to untreated patients. Increases in pre-existing immune responses to tumor protein correlate with better clinical outcomes. Such immune responses generally can be assessed using standard propagation, cytotoxicity, or cytokine assays, which can be performed using samples obtained from the patient before and after treatment.

The invention will be further described by means of the following non-limiting examples and research examples which are given for illustrative purposes only.

Examples

P r z k ł a d 1

Modifications of acyl chains of primary fatty acids

This example describes the preparation of primary fatty acid derivatives which have acyl chains of primary fatty acids of variable length, either alone or in combination with variable secondary fatty acid chains, for example compounds 1a-c and 2a-c in which the primary fatty acids with a short (C6) and medium (C10) chain are combined with short, medium or long chain secondary fatty acids.

These compounds are prepared using either the well-established serine aglycon _(R1 = CO ₂ H) or, alternatively, using a chemically more stable and ionizable serinol phosphate aglycon unit (R1 = CH2OPO3H2). The choice of serine / serinol phosphate will be based on comparing the biological activities of the known seryl derivatives 3a, b with the new serinol phosphates 4a, b.

PL 220 536 B1

3a _R2 = C6 acyl (RC-526) 3b R ₂ = C ₁₀ acyl (RC-527)

4a R2 = Cgacyl 4b R2C10 acyl

Derivatives of the original chain modified compounds are synthesized by modification of the previously described method (B1 in Johnson et al., US Patent No. 6,355,251) using a common advanced intermediate that allows the introduction of amide or ester bound acyloxy acids at the end of the synthesis (Scheme I). The initial step in the synthesis consists in the glycosylation of acceptor 6 with the known tetraacetate 5 (obtained in 4 steps from glucosamine) to obtain β-glycoside 7 and conversion of 7 into a common advanced intermediate (CAI) 8 which is optimized for R1 = CO2Bn. Selective 4-O-acylation and N-deprotection / acylation give the hexaacyl derivative 9 which is converted in 3-4 steps in 1a-c or 2a-c by phosphorylation and deprotection.

The required (R) -3-n-alkanoyloxyalkanoic acids are obtained according to Keegan et al., Tetrahedron; Asymmetry, 7 (12): 3559-3564, 1996, starting from the corresponding 3-oxomethyl esters. The high chemical and diastereomeric purity of products 1 and 2 is achieved either by normal phase chromatography on silica gel or, alternatively, by cellulose chromatography or liquid-liquid partition chromatography on Sephadex LH-20. Purity 13 wydzielo1 salts triethyl determined by spectroscopic means (IR, ¹ H and ¹³ C NMR) and physical (combustion analysis by FAB-MS) and HPLC.

P r z k ł a d 2

Glycyl and phosphonooxyethyl (PE) compounds

This example describes the synthesis of glycyl compounds 11a, b and phosphonooxyethyl (PE) compounds 12a, b which are almost regioisomeric from 3a, b and 4a, b.

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12a R ₂ = Caacy! 12b R ₂ = C ₁₀ acyI lia R ₂ = Ceacyl lib R ₂ = C ₁₀ acyl

These compounds are most readily obtained by a more convergent synthesis than the synthesis depicted in Scheme I, in which a common C6- or C10-glycosyl donor 13 is coupled to an appropriate N-acylated (or alternatively an N-Troc-protected, not shown) acceptor unit 14 or 15. in the presence of silver ion to give β-glycosides 16 (Scheme II). Glycine 14 acceptor is prepared according to Bulus et al., J Med Chem, 35 (19): 3463-3469,1992, from ethanolamine and benzyl (or t-butyl) bromoacetate followed by N-acylation or protection. Phosphate 15 is obtained by monophosphorylation of N-acylated (or protected) diethanolamine. N-removal of the protecting group / acylation or N, N-diacylation of a Trocrotected aglycone (Jiang et al., Tetrahedron, 58 (43): 8833-8842, 2002) of 16 β-glycosides and cleavage of phenyl and other protecting groups is expected the resulting hexaacylated derivative 17 will give the desired compounds 11a, b and 12a, b, which are isolated and characterized as their triethylammonium salts after chromatographic purification over silica gel or LH-20 gel or DEAE cellulose.

Compounds 16 are novel compounds and form yet another aspect of the disclosure.

P r z k ł a d 3

Secondary ether lipids

This example describes the synthesis of (R) -3-alkyloxytetradecanoic acid derivatives (18a, b) that are resistant to unfavorable metabolism and / or aqueous hydrolysis. For the synthesis of compounds 18a, b, first the ether lipid analogs of the secondary fatty acid compounds 3a, b or the corresponding serinol phosphates 5a, b must be obtained. As shown retrosynthetically in Scheme III, the synthesis of the target molecules 18a, b can be achieved by acid substitution (R) -3 -hexyloxytetradecanoic acid or (R) -3-decyloxytetradecanoic acid instead of the corresponding

GB 220 536 B1 acyloksykwasów, starting from 3-selective O-acylation of common advanced intermediate 8 in Scheme I and proceeding through intermediate 9 _(R2 = C ₆ - or C ₁₀ -alkyl, n = 9). The required 19 alkyloxy acids are synthesized from (R) -3-hydroxytetradecanoic acid or its phenacyl ester, intermediates in the synthesis of acyloxy acids, by known methods with an overall yield of> 50% (Keegan et al., Tetrahedron: Asymmetry, 7 (12): 3559-3564 , 1996, Watanabe et al., Carbohydr Res, 332 (3): 257-277, 2001, Jiang, Bioorg Med Chem Lett, 12 (16): 2193-2196, 2002, Christ et al., U.S. Patent No. 5,530,113, 1996).

S RjoCHaCan or COjBn Joint Advanced Intermediate

18a π-t (O; łkil)

18b n = S (C 10 aM) R 3 CH 10 H J or CO 2 H

ΗΗΓπκ ΝΗΤπκ

Scheme III

P r z k ł a d 4

Primary and secondary ether lipids

This example describes compounds (20a, b) containing a primary ether lipid in the C-3 position of the sugar and three secondary ether lipids. These compounds are synthesized by the alkylation of acetonide 21, an intermediate in the synthesis of glycosyl donor 13 (Scheme II) with sulfonate 22, which in turn is prepared in one step from alcohol precursor 19 to give the diether 23 (Scheme IV). 4,6-functional groups and anomeric activation yield glycosyl chloride 24, which is then processed as in Scheme II using the appropriate alkyloxy acids in the N-acylation steps. ^{In an alternative scheme, the 2-azido derivative 42} or the 2-tri-fluoroacetamido ^{derivative 45} may be used in the 3-O-alkylation step.

P r z k ł a d 5

C-6-modified compounds

This example describes compounds that are blocked with the 6-hydroxy. In this example, methyl ether or a fluoro group is used in combination with compounds 25a, b and 26a, b of either seryl phosphate or serinol. As mentioned above, these compounds also form an aspect of the disclosure.

PL 220 536 B1

Compounds are prepared from diol 27 as shown in Scheme V. Intermediate 27, obtained in two steps from acetonide 21, is functionalized at the 6-position by known methods (Christ et al., US Patent No. 5,530,113, 1996, Watanabe et al., Carbohydr Res, 333 (3): 203-231, 2001) to give alcohol 28. Conversion of 28 to chloride 29 in two steps and treatment according to Scheme II yields target molecules 25a, b and 26a, b. Compounds with primary and / or secondary ether linkages as described above in Example 4, may be modified as described in this example to further protect the molecules from chemical and enzymatic degradation.

Claims (5)

1. Compound of formula: wherein n is 1 or 5 and R ₆ is COOH or CH ₂ OPO ₃ H ₂ . 2. A compound according to claim The method of claim 1, wherein n is 1. 3. The compound according to p. The method of claim 1, wherein n is 5. 4. The compound according to p. The process of claim 2, wherein R6 is COOH. 5. The compound according to p. 3. The process of claim 3, wherein R6 is COOH. Publishing Department of the PPO